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Article (refereed) - postprint Knudsen, F.R.; Pottinger, T.G.. 1999 Interaction of endocrine disrupting chemicals, singly and in combination, with estrogen-, androgen-, and corticosteroid-binding sites in rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology, 44 (3). 159-170. 10.1016/S0166-445X(98)00079-4

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1

Interaction of endocrine disrupting chemicals, singly and in combination, with

estrogen-, androgen-, and corticosteroid-binding sites in rainbow trout (Oncorhynchus

mykiss)

F. R. Knudsen and T. G. Pottinger*

Department of Biology, University of Oslo, PB 1066, Blindern, N-0316 Oslo, Norway.

*NERC Institute of Freshwater Ecology, Windermere Laboratory, The Ferry House,

Ambleside, Cumbria, LA22 0LP, United Kingdom.

Corresponding author (present address):

Frank Reier Knudsen

Simrad AS

P.O. Box 111

N-3191 Horten

Norway

Telephone: +47 3303 4253

Fax: +47 3304 2987

E-mail: frank.reier.knudsen@simrad.no

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Abstract

The ability of chemicals with known endocrine disrupting activity to interact with three major

teleost steroid-binding sites was evaluated. Representative alkylphenols and phthalates, the

pesticides dieldrin and toxaphene, the mycoestrogen zearalenone and the phytoestrogen

genistein were tested for their ability to displace native ligand from putative estradiol receptor

(ER), testosterone receptor (TR) and cortisol receptor (CR) from rainbow trout liver and

brain. The ER displayed a higher affinity for alkylphenols than for phthalates, but both groups

of compounds were 104-2.105 times less potent than estradiol in displacing specifically bound

[3H]estradiol. The displacement of bound [3H]estradiol did not increase when these

compounds were tested in combination. Toxaphene and dieldrin did not bind to the trout ER,

either alone or in combination. Zearalenone and genistein were about 103-fold less potent

than estradiol in displacing specifically bound [3H]estradiol from the trout ER and showed no

increase in potency when tested in combination. None of the compounds tested showed

evidence of binding to the TR or the CR, failing to displace specifically bound

[3H]testosterone and [3H]cortisol respectively. It is concluded that the compounds tested are

exclusively estrogenic in rainbow trout, albeit weakly so, and do not display any synergistic

effects.

Keywords: Endocrine disruptors; rainbow trout; estradiol receptor; androgen receptor;

cortisol receptor; synergism.

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1. Introduction

Many xenobiotics have been demonstrated to have a weakly estrogenic effect in fish (Thomas

& Smith, 1993; Jobling et al., 1995; Sumpter and Jobling, 1995; Donohoe and Curtis; 1996;

Jobling et al., 1996). These chemicals bind to the fish ER (Thomas and Smith, 1993; White et

al., 1994; Jobling et al., 1995), stimulate expression of the vitellogenin gene and synthesis of

vitellogenin in fish hepatocytes in vitro (Jobling and Sumpter, 1993; Pelissero et al., 1993),

and induce hepatic production of vitellogenin and zona radiata proteins in vivo (Jobling et al.,

1996; Arukwe et al., 1997; Harries et al., 1997). Among the most widely distributed of these

chemicals in the aquatic environment are alkylphenols, phthalates, metabolites of

dichlorodiphenyltrichloroethane (DDT), polychlorinated biphenyls (PCBs) and various

pesticides. In particular, relatively high concentrations of alkylphenols and phthalates have

been measured in treated sewage effluent (Giger et al., 1984; Giger et al., 1987; Lahl et al.,

1988; Blackburn and Waldock, 1995; Petrosky and Vidic, 1996; Vitali et al., 1997) and it is

well documented that exposure to treated sewage and waste water from oil industry processes

induces the production of vitellogenin and zona radiata proteins in fish (Purdom et al., 1994;

Arukwe et al., 1997; Harries et al., 1997; Knudsen et al., 1997). Although recent data

suggest that at least some of the estrogenicity of sewage effluent may be ascribed to steroids

of human origin (Anon, 1997) there is no doubt that an assemblage of potentially estrogenic

chemicals occurs in such effluents. Therefore, evaluating the effect of chemicals in

combination, rather than in isolation, is the most realistic approach to assessing

environmental impact. Sumpter and Jobling (1995) observed a more than additive effect of a

mixture of estrogenic chemicals on the production of vitellogenin by fish hepatocytes in vitro,

suggestive of a synergistic effect. The possibility of synergistic relationships between

estrogenic chemicals is a topic that has raised much concern and debate recently. Following

the publication and subsequent withdrawal of a paper purporting to document very marked

4

synergistic effects (Arnold et al., 1996; McLachlan, 1997) there is confusion as to whether

synergism between estrogenic chemicals exists or not (Arnold et al., 1997; Ashby et al., 1997;

Ramamoorthy et al., 1997). However, only a few chemicals have been tested for synergistic

effects, and many of the environmentally abundant xenoestrogens have not been screened for

such actions.

Interest to date, in both mammals and lower vertebrates, has focused on the interaction of

endocrine disrupting chemicals with the estrogen receptor system. Only limited effort has

been directed towards assessing the potential disruption of other endocrine axes (e.g. Kelce et

al., 1995; Kelce and Wilson, 1997) despite the fact that these are potentially vulnerable to

interference by xenobiotics.

The aims of the present study were twofold: to compare the relative binding affinity of a

range of estrogenic chemicals for the trout hepatic estrogen receptor, and to investigate

whether the ER binding affinity increases when these chemicals are present in combination;

secondly, to determine whether chemicals already established to possess estrogenic activity

can bind to the trout androgen and corticosteroid receptor.

2. Materials and methods

2.1 Chemicals tested

The chemicals used in this study were obtained from the following sources: phenol (Fluka);

4-n-alkylphenols and butylphenol (Kebo); pentylphenol (Arcos); hexylphenol (TCL);

heptylphenol (Eastman); octylphenol (Aldrich); nonylphenol (Arcos); dodecylphenol (Arcos);

5

butylbenzylphthalate (TCL); all other phthalates (Sigma); dieldrin (Sigma); toxaphene

(Aldrich); p,p’-DDT (Aldrich); genistein and zearalenone (Sigma); 17-estradiol,

testosterone, cortisol (Sigma). All radiolabelled compounds were obtained from Amersham

Life Science.

2.2 Fish

Mixed-sex rainbow trout (400-600g, Stirling strain) were used throughout these studies.

Stock fish were maintained in 1500 l outdoor fiberglass tanks, each supplied with a constant

flow of lake water (25 l/min.) at 6 C and were fed thrice-weekly with commercial trout feed

(Trouw Aquaculture; 1% body weight/day).

2.3 Tissue preparation

The interaction of the selected xenobiotics with testosterone-binding sites was assessed using

cytosol prepared from whole brain homogenates, while the assessment of interactions with

estrogen and corticosteroid binding were evaluated using liver cytosol. The procedures

employed for tissue preparation and the characterisation of specific estrogen and

corticosteroid binding in rainbow trout liver cytosols has been fully reported previously

(Pottinger, 1990; Pottinger and Pickering, 1990). Testosterone binding in whole brain cytosol

was measured using the techniques previously described for the quantification of androgen

binding in trout olfactory tissue (Pottinger and Moore, 1997) and was fully characterised in

terms of affinity, specificity and capacity (Johnsen, Pottinger and Knudsen, unpublished).

6

2.4 Competition study

Aliquots (200 l) of cytosol were incubated in triplicate together with 1 pmol of labelled

steroid ([2, 4, 6, 7-3H] estradiol, 2, 4, 6, 7-3H]cortisol, or 2, 4, 6, 7-3H testosterone;

Amersham) together with an increasing concentration of competitor, in both the presence

(non-specific binding) and absence (total binding) of a 1000-fold excess of inert steroid, for 4

h at 4C. When chemicals were tested in combination the concentration of the chemicals was

halved to obtain the same total molarity in all experiments. After incubation, unbound steroid

was removed by the addition to each tube of 200 l of ice-cold dextran-coated charcoal

suspension, followed by incubation on ice for 10 min. and centrifugation at 1000g for 10 min.

at 4C. Aliquots of supernatant (300 l) were added to 5 ml of scintillant (Ecoscint A,

National Diagnostics) in a vial, and counted under standard tritium conditions. Specific

binding was calculated from the difference between total and non-specific binding and

corrected for protein concentration.

2.5 Protein determination

Protein concentration in the various preparations was determined according to the method of

Ohnishi and Barr (1978).

3. Results

Results are presented as means of triplicate incubations. Standard errors are too small to be

depicted on the figures. The relative potency of the chemicals tested is referred to as the ratio

between the amount of steroid and amount of competitor required to produce the same

percentage displacement of the 3Hsteroid.

7

All the alkylphenols tested were found to bind to the ER receptor with a relatively low

affinity (Fig. 1.). Concentrations of alkylphenols approximately 104-fold greater than those of

estradiol were required to produce similar amounts of displacement of specifically bound

[3H]estradiol. The maximum displacement achieved was between 40-80% when the

concentration of alkylphenols were approximately 50 nmols/tube (165 M). Higher

concentrations of alkylphenols were not tested.

Phthalates also displaced 3Hestradiol from the ER, but higher concentrations were required

than for the alkylphenols (Fig. 2.). A concentration of phthalates approximately 2.105-fold

greater (50 nmols/tube; 165 M) than that of estradiol was required to produce an equivalent

10-25% displacement of specifically bound 3Hestradiol. The relative potency of both

alkylphenols and phthalates in displacing 3Hestradiol showed considerable variation with

concentration of the competitor. For this reason, no attempt was made to rank the chemicals

in terms of their relative estrogenicity.

When several combinations of alkylphenols and phthalates were employed in the assay

system (OP+BBP, PP+BBP, OP+DEHP, nonylphenol+DEP, hexylphenol+DAP), no increase

in the ability of the chemicals to displace specifically bound 3Hestradiol was observed,

implying an absence of synergistic effects. The results from experiments in which a

combination of OP and BBP, and PP and BBP were tested are presented (Figs. 3a and 3b,

respectively).

8

Two pesticides, toxaphene and dieldrin, were tested for binding to the ER individually and in

combination. Neither chemical, alone or in combination, displaced specifically bound

3Hestradiol (Fig 4.).

In contrast, the phytoestrogen genistein and the mycoestrogen zearalenone did displace

specifically bound 3Hestradiol, displaying a higher affinity for the binding site than the

alkylphenols and phthalates (Fig. 5). A 40-60% displacement of 3Hestradiol was achieved at

a concentration about 103-fold the estradiol concentration required to achieve the same level

of displacement. No evidence of synergism was observed when these chemicals were tested

in combination.

All the chemicals were tested for their ability to displace specifically bound 3Htestosterone

in brain cytosol. No displacement of testosterone was observed at concentrations of

competitor up to 104 times the concentration of 3Hligand. Representative results from the

testing of DBP, BBP, PP, OP, dieldrin and p,p’- DDT are shown in Fig. 6.

In addition, all chemicals were assessed for their ability to displace specifically bound

3Hcortisol in trout liver cytosol. No displacement of cortisol was found even at a

concentration of competitor 5.104-fold that of 3Hcortisol. At high concentrations of the

chemicals (>5.103-fold that of the ligand, equivalent to 5 nmols/tube (16 M) an apparent

increase in binding to more than 100% of the initial level observed in the absence of

competitor was noted (see discussion). Representative results obtained from testing of PP,

OP, DBP, BBP and DEHP are shown in Fig. 7.

9

4. Discussion

The results of these studies demonstrate that alkylphenols and phthalates bind to the ER of

rainbow trout liver with a relatively low affinity. The alkylphenols were between 5.103-5.104

times less potent than estradiol. This is in agreement with the results of previous studies

which utilised trout ER (Sumpter and Jobling, 1995). In the present study the phthalates were

found to be as much as 2.105 times less potent than estradiol in displacing specifically bound

3Hestradiol, an observation consistent with previous reports (Jobling et al., 1995). However,

in the present study BBP showed a similar level of competitiveness to the other phthalates

tested whereas others have observed BBP to be a more potent competitor (Jobling et al.,

1995; Harris et al., 1997). We cannot explain this disagreement but the source and/or

formulation of the BBP used in the studies may be a factor.

Using a recombinant yeast estrogen receptor screen Routledge and Sumpter (1997) were able

to rank alkylphenols with respect to their estrogenicity and to demonstrate the importance of

certain structural features in determining estrogenicity. Phthalates were not tested. These

authors noted differences in estrogenicity among the alkylphenols of a greater magnitude than

was observed in the present study and found these differences to be consistent independent of

concentration This contrasts with the present study in which the relative estrogenicity of the

alkylphenols and phthalates was observed to change with concentration and suggests that the

recombinant yeast screen is a system of greater resolution than the in vitro hepatic cytosol

receptor assay. The contrasting results obtained using the two assay systems may reflect the

possibility that although ER binding affinity among the alkylphenols is similar, the ability of

specific chemicals to activate the receptor or associate the ER-ligand-complex to the estrogen

response element on the DNA, may be different. However, both these test systems are

artificial and neither necessarily reflects the situation in vivo. We have recently screened a

10

range of alkylphenols for estrogenicity in a fish hepatocyte culture (unpublished results) and

found that the range of potency is less than that reported by Routlege and Sumpter (1997) and

more akin to that observed in the trout hepatic ER assay. Compared to both the hepatic

cytosol receptor assay and the yeast screen, a hepatocyte culture more closely resembles the in

vivo situation because it does facilitate at least some of the metabolic transformations

undertaken by the liver (Pelissero et al., 1993).

The data presented by Arnold et al. (1996) evoked much speculation regarding the possibility

that synergism occurs among estrogenic chemicals, and doubts have been raised that such

synergism does occur (Ashby et al., 1997; McLachlan, 1997; Ramamoorthy et al., 1997). The

potential for synergistic actions between alkylphenols and phthalates has not previously been

tested in assays based on the hepatic ER. In the present study, no evidence of synergistic

effects on binding to the ER was observed when alkylphenols and phthalates were combined.

Nor was binding of dieldrin and toxaphene to the trout hepatic ER observed, either alone or in

combination, despite a report that these environmentally persistent (Paasivirta and Rantio,

1991; Stern et al., 1996) insecticides interact with the human ER synergistically (Arnold et

al., 1996). However, it must be noted that this result was not found to be reproducible

(McLachlan, 1997). It is therefore reasonable to assume that these compounds will not cause

estrogenic effects in fish. This is consistent with the findings of others who have failed to

observe synergistic interactions between these chemicals (Ramamoorthy et al., 1997; Ashby

et al., 1997). However, Arnold et al. (1997) have reported that a combination of toxaphene

and dieldrin bind to the alligator ER in a synergistic manner. These contradictory results

require clarification and underline the need for a unifying and reliable method to determine

the estrogenicity of chemicals in vertebrate systems.

11

The binding of genistein and zearalenone to the trout hepatic ER was also assessed. Both

these compounds are abundant in many food sources (Livingston, 1978; Verdeal and Ryan,

1979; Kuiper-Goodman et al., 1987), bind to the human ER with a relatively high affinity

(Miksicek, 1994), and stimulate growth in estrogen sensitive cell lines (Mayr et al., 1992).

Phytoestrogens are also found in fish diet, both of commercial and natural origin, and could

thus exert estrogenic effects on fish (Pelissero and Sumpter, 1992). We found that

zearalenone and genistein bound to the ER with a higher affinity than the alkyphenols and the

phthalates, displaying an apparent affinity of approximately 103-fold less than that of

estradiol. This is similar to the binding affinity previously reported for these compounds in

trout and human ER assays (Miksicek, 1994; Sumpter and Jobling, 1995) and suggests that

these compounds could exert more profound estrogenic effects than alkylphenols and

phthalates. However, as for the other chemicals tested in this study, no evidence for a

synergistic increase in binding affinity for the ER was found when testing these compounds in

combination.

None of the compounds tested was successful in displacing [3H]testosterone from a specific

binding site in trout brain cytosol suggesting that these chemicals are not likely to manifest

androgenic or antiandrogenic effects in fish. Previously it has been reported that p,p’-DDT

binds to the TR from rat prostate with a relatively high affinity (Kelce et al., 1995). In the

present study p,p’-DDT did not bind to the putative TR from fish brain suggesting that

p,p’-DDT is not androgenic in fish. These contrasting observations may reflect species

differences in the ligand specificity of the TR, similar to those reported for the binding of

other xenoestrogens to steroid receptors. DDT and PCB metabolites displayed no binding to

the ER from spotted seatrout (Cynoscion nebulosus; Thomas and Smith, 1993) although the

same chemicals have been demonstrated to bind to a mammalian ER (McLachlan et al.,

12

1984). Steroid receptor structure varies between animals. For example, the fish ER (Pakdel et

al., 1989, 1990) contains fewer amino acids than the human ER (Green et al., 1986) although

the hormone binding site is conserved (Pakdel et al., 1990; Le Roux et al., 1993). It has been

suggested that endocrine disruptors can affect receptor activation through binding to sites on

the steroid receptor other than the hormone binding site (McLachlan and Arnold, 1996) and if

this is the case, species differences in binding of chemicals to steroid receptors is likely.

Furthermore, the rainbow trout ER has a lower affinity for estradiol than the human ER (Le

Drean et al. 1995; Petit et al., 1995) which may also give rise to differences in ligand activity

in different test systems.

To our knowledge, there have been no studies which have examined the binding of

xenobiotics to the CR. However, this receptor system is potentially of some significance with

regard to the potential impact of endocrine disrupting chemicals. It has been suggested that

exposure of animals to endocrine disruptors can lead to immunosuppression (see Kavlock et

al., 1996), but the mechanism underlying this effect is unclear. Cortisol is a well-established

immunosuppressant in vertebrates (Parillo and Fauci, 1979; Bateman et al., 1989) including

fish (Kaatari and Tripp, 1987; Tripp et al., 1987; Pickering and Pottinger, 1989). The effect of

cortisol on the immune system in fish is most likely mediated via a CR (Maule and Schreck,

1990) and therefore susceptible to interference. However, none of the chemicals tested in the

present study manifested any affinity for the trout hepatic CR. Nonetheless, the possibility

that there are classes of xenobiotic compounds which do interact with the CR cannot be

excluded and is worthy of further investigation. Such considerations have to a large extent

been overshadowed by the emphasis place on estrogenic effects in current efforts in this field.

13

A phenomenon occasionally observed in the present study was an apparent increase in

binding of labelled cortisol at high concentration of competitor (see Fig. 7). This may be an

artefact arising due to the hydrophobic nature of many of the chemicals tested. If at high

concentrations the critical micelle concentration (CMC) of these chemicals is reached,

micelle formation will occur, with a resulting reduction in the amount of chemical available

for exchange with the receptor. Micelle formation could theoretically “upset” the assay in

different ways giving rise to spurious results, for example, by “protecting” [3H]steroid from

adsorption to the dextran-coated charcoal used to separate bound ligand from free. From our

results, it is not possible to provide an explanation for the mechanism behind this artefact.

Conclusion

In conclusion, alkylphenols and phthalates have a low affinity for the trout hepatic ER and

should therefore be classified as weak estrogens in trout. No increase in their estrogenicity is

apparent when they are in combination, suggesting that synergistic effects are absent. Dieldrin

and toxaphene appear not to be estrogenic to fish either alone or in combination. Zearalenone

and genistein are more estrogenic than the alkylphenols and phthalates, and are more likely

than these compounds to exert estrogenic effects in vivo. None of the chemicals tested were

found to bind to putative receptors for testosterone and cortisol and it is therefore unlikely

that they will exert disruptive effects on processes normally mediated by these hormones.

However, this does not exclude the possibility that other classes of compounds have

androgenic or corticosteroidogenic effects. In addition, it should be borne in mind that a

receptor assay does not take into account metabolism of the chemicals tested, so before final

conclusions are made, in vivo screening should be performed.

14

Acknowledgements

This work was financially supported by the Norwegian Research Council and the Bellona

Foundation. T.G.P. was funded by the Natural Environment Research Council (UK).

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Figure legends

Fig. 1. Displacement of 3Hestradiol from the trout hepatic ER by a range of

4-n-alkylphenols. The data are presented as the percentage of bound 3Hestradiol for the

given concentration of competitor. Hepatic cytosol was incubated with 1 pmol 3Hestradiol

together with either one of a range of alkylphenols (500, 5000 and 50000-fold the

concentration of 3Hestradiol) or inert estradiol (0.5, 5, 50 and 500-fold the concentration of

3Hestradiol). The competitors used were phenol, butylphenol (butyl), pentylphenol

(pentyl), hexylphenol (hexyl), heptylphenol (heptyl), octylphenol (octyl), nonylphenol (nonyl)

and dodecylphenol (dodecyl).

Fig. 2. Displacement of 3Hestradiol from the trout hepatic ER by a range of phthalates.

Experimental conditions were as described for Fig. 1. The phthalates tested were Bis

(2-etylhexyl) phthalate (DEHP), diallylphthalate (DAP), dinonylphthalate (DNP),

butylbenzylphthalate (BBP), dibutylphthalate (DBP) and dietylphthalate (DEP).

Fig. 3. Displacement of 3Hestradiol from the trout hepatic ER by (a) pentylphenol (PP),

butylbenzylphthalate (BBP) and PP+BBP in combination, and (b) octylphenol (OP) and bis

(2-etylhexyl) phthalate (DEHP) and OP+DEHP in combination. Note that the concentration

of each of the chemicals when tested in combination was halved. Experimental conditions

were as described for Fig. 1.

Fig. 4. Displacement of 3Hestradiol from the trout hepatic ER by toxaphene, dieldrin and

toxaphene and dieldrin in combination (diel + tox). Note that the concentration of each of the

23

chemicals when tested in combination was halved. Experimental conditions were as described

for Fig. 1.

Fig 5. Displacement of 3Hestradiol from the trout hepatic ER by genistein, zearalenone and

genistein and zearalenone in combination (Gen + Zer). Note that the concentration of each of

the chemicals when tested in combination was halved. Experimental conditions were as

described for Fig. 1.

Fig. 6. Displacement of 3Htestosterone from the high affinity testosterone binding site in

rainbow trout brain cytosol by dibutylphthalate (DBP), butylbenzylphthalate (BBP),

4-n-pentylphenol (PP), 4-n-octylphenol (OP), dieldrin and p,p’ DDT. Experimental

conditions were as described for Fig. 1. except that inert testosterone was employed.

Fig. 7. Displacement of 3Hcortisol from the high affinity cortisol binding site in rainbow

trout liver cytosol by pentylphenol (PP), octylphenol (OP), dibutylphthalate (DBP),

butylbenzylphthalate (BBP) and bis (2-etylhexyl) phthalate (DEHP). Experimental conditions

were as described for Fig. 1. except that inert cortisol was employed.

24

Figure 1.

X [competitor]

%

bin

din

g

0

20

40

60

80

100

120

Estradiol Phenol Butyl Pentyl Hexyl Heptyl Octyl Nonyl Dodecyl

0 5 50 500 5000 50000 0,5

F Figure 2.

X [competitor]

% b

indin

g

-20

0

20

40

60

80

100

120

Estradiol DEHP DAP DNP BBP DBP DEP

5 50 500 50000 0,5 5000 0

25

Figure 3(a)

X [competitor]

% b

indin

g

0

20

40

60

80

100

120

Estradiol PP BBP PP+BBP

0 0,5 5 50 500 5000 50000

Figure 3(b)

X [competitor]

% b

indin

g

0

20

40

60

80

100

120

Estradiol OP DEHP OP+DEHP

0 0,5 5 50 500 5000 50000

26

Figure 4.

X [competitor]

% b

indin

g

-20

0

20

40

60

80

100

120

Estradiol Dieldrin Toxaphene D+T

0 1 10 100 1000 10000 100000 0,1

Figure 5.

X [competitor]

% b

indin

g

0

20

40

60

80

100

120

Estradiol Genistein Zearalenone Gen+Zer

0 1 10 100 1000

27

Figure 6.

X [competitor]

% b

indin

g

0

20

40

60

80

100

120 Testosteron DBP BBP PP OP dieldrin p,p' DDT

0 0,1 1 10 100 1000 10000

Figure 7.

X [competitor]

% b

indin

g

-50

0

50

100

150

200

250

Cortisol PP OP DBP BBP DEHP

0 0,5 5 50 500 5000 50000